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Protein N-methylation has recently emerged as a major mechanism involved in regulating gene transcription and epigenetic inheritance. Transcriptional coactivators and corepressors have both been shown to associate with protein N-methyltransferases. These enzymes methylate arginine or lysine residues within histones as well as other proteins, such as transcription factors. Here, we discuss the recent...
Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction...
The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established. Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications. Antibodies against acetylated...
It is becoming clear that the structure of cellular chromatin is dynamic and capable of undergoing rapid changes to respond to the metabolic requirements of the cell. These changes have a direct impact on gene expression and, therefore, the chromatin context must be considered when biochemical reactions that involve DNA are studied. Over the past several decades, a number of methods for assembling...
There is much evidence indicating the importance in gene regulation of the positions of nucleosomes with respect to DNA sequence. Low resolution chromatin structures have been described for many genes, but there is a dearth of detailed high resolution chromatin structures. In the cases where they are available, high resolution maps have revealed much more complex chromatin structures, with multiple...
The use of fission yeast as a model system for studies of chromosome biology has contributed to several key advances in the last few years. The structure of its large complex centromeres and composition of its transcriptionally silent heterochromatin resemble those of metazoa. The application of chromatin immunoprecipitation to fission yeast has been instrumental in these advances and we describe...
Until recently, identifying the specificities of enzymes that post-translationally modify core histones was performed in vitro using synthetic peptides, purified mononucleosomes or short nucleosome arrays. Unfortunately, the variable results obtained for identical enzymes are often dependent on the in vitro conditions employed. These results are consistent with the conclusion that the manner in which...
The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.
The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation...
The core histone tail domains play important roles in different stages of chromatin condensation. The tails are required for folding nucleosome arrays into secondary chromatin structures such as the ∼30nm diameter chromatin fiber and for mediating fiber–fiber interactions important for formation of tertiary chromatin structures. Crosslinking studies have demonstrated that inter-nucleosomal tail–DNA...
Macromolecular assemblies and machines undergo large-scale conformational changes as essential features of their normal function. Modern stopped-flow instrumentation and biotechnology combine to provide a powerful tool for characterizing the rates and natures of these conformational changes. Standard commercially available instruments provide extraordinary sensitivity and speed, allowing analysis...
Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since...
Combinations of phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones comprise what is referred to as the “histone code”. These marks influence processes from transcription to DNA replication, where gaining access to DNA organized in chromatin is necessary. Much emphasis has been placed on the role of histone ubiquitylation and sumoylation during the process of transcription...
Epigenetic control of genome function is an important regulatory mechanism in diverse processes such as lineage commitment and environmental sensing, and in disease etiologies ranging from neuropsychiatric disorders to cancer. Here we report a robust, high-throughput targeted, quantitative mass spectrometry (MS) method to rapidly profile modifications of the core histones of chromatin that compose...
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